Eisen.Geba.Jgi2009b

GEBA A genomic encyclopedia of bacteria and archaea Jonathan A. Eisen JGI User Meeting 2009

“ Nothing in biology makes sense except in the light of evolution.” T. Dobzhansky (1973)

rRNA Tree of Life

The Tree is not Happy

From http://genomesonline.org

At least 40 phyla of bacteria
Acidobacteria Bacteroides Fibrobacteres Gemmimonas Verrucomicrobia Planctomycetes Chloroflexi As of 2002 Based on Hugenholtz, 2002 Proteobacteria Chlorobi Firmicutes Fusobacteria Actinobacteria Cyanobacteria Chlamydia Spriochaetes Deinococcus-Thermus Aquificae Thermotogae TM6 OS-K Termite Group OP8 Marine GroupA WS3 OP9 NKB19 OP3 OP10 TM7 OP1 OP11 Nitrospira Synergistes Deferribacteres Thermudesulfobacteria Chrysiogenetes Thermomicrobia Dictyoglomus Coprothmermobacter

Acidobacteria Bacteroides Fibrobacteres Gemmimonas Verrucomicrobia Planctomycetes Chloroflexi
Genome sequences are mostly from three phyla
As of 2002 Based on Hugenholtz, 2002 Proteobacteria Chlorobi Firmicutes Fusobacteria Actinobacteria Cyanobacteria Chlamydia Spriochaetes Deinococcus-Thermus Aquificae Thermotogae TM6 OS-K Termite Group OP8 Marine GroupA WS3 OP9 NKB19 OP3 OP10 TM7 OP1 OP11 Nitrospira Synergistes Deferribacteres Thermudesulfobacteria Chrysiogenetes Thermomicrobia Dictyoglomus Coprothmermobacter

Some other phyla are only sparsely sampled

Same trend in Archaea

Need for Tree Guidance Well Established
Common approach within some eukaryotic groups
Many small projects funded to fill in some bacterial or archaeal gaps
Phylogenetic gaps in bacterial and archaeal projects commonly lamented in literature

Solution I: sequence more phyla
NSF-funded Tree of Life Project
A genome from each of eight phyla
Eisen, Ward, Badger, Wu, Wu, et al. Proteobacteria Chlorobi Firmicutes Fusobacteria Actinobacteria Cyanobacteria Chlamydia Spriochaetes Deinococcus-Thermus Aquificae Thermotogae TM6 OS-K Termite Group OP8 Marine GroupA WS3 OP9 NKB19 OP3 OP10 TM7 OP1 OP11 Nitrospira Synergistes Deferribacteres Thermudesulfobacteria Chrysiogenetes Thermomicrobia Dictyoglomus Coprothmermobacter

Bacterial aTOL Project AIMS
Improve resolution of deep branches in the bacterial tree
Launch biological studies of these phyla and discover functional novelty
Leverage data for interpreting environmental surveys

T. roseum genome

The Tree of Life is Still Angry

Within Phyla Diversity Immense
Each phyla represents billions of years of evolution
Some have hundreds of major lineages
New lineages are being discovered all the time
Most branches within most phyla have few or no genomes

Major Lineages of Actinobacteria

Additional Impetus for Tree Guided Projects
Suggestion to sequence all bacteria and archaea in Bergey’s Manual (Stevens et al)
Success in sequencing genomes from across the tree in animals
Multiple government reports suggest a more systematic approach to sequencing is needed

Most phyla with cultured species are sparsely sampled
Lineages with no cultured taxa even more poorly sampled
Solution - use tree to really fill gaps
Well sampled phyla Proteobacteria Chlorobi Firmicutes Fusobacteria Actinobacteria Cyanobacteria Chlamydia Spriochaetes Deinococcus-Thermus Aquificae Thermotogae TM6 OS-K Termite Group OP8 Marine GroupA WS3 OP9 NKB19 OP3 OP10 TM7 OP1 OP11 Nitrospira Synergistes Deferribacteres Thermudesulfobacteria Chrysiogenetes Thermomicrobia Dictyoglomus Coprothmermobacter

http://www.jgi.doe.gov/programs/GEBA/pilot.html

GEBA Pilot Project Overview
Select 200 organisms using tree
Develop high throughput pipeline for strain growth and DNA preparation
Sequence and finish 100
Annotate, analyze, release data
Assess benefits of tree guided sequencing

GEBA Pilot I: Selecting Targets

GEBA Pilot II: The Importance of Project Management

GEBA Project Flowchart
GEBA Proposal Scientific and Technical Review 1 Negotiate Scope of Work Receive Starting Material 1 OK? Project Initiation Sequencing Annotation Draft Sequencing and Assembly 1 Finish Sequencing and Assembly 2 IMG 1 Finish Annotation 3 Complete Genome GenBank Submission 1 Draft Annotation 3 Shotgun Genome GenBank Submission 1 IMG – ER 1 1 PGF 2 LANL 3 ORNL OK? OK? IMG – ER 1 Gene-QA 1 David Bruce, Lynne Goodwin et al

GEBA Pilot III: Partnership with DSMZ

GEBA Biggest Challenge: Getting DNA
Getting quality DNA is biggest bottleneck
Solution: Beg Borrow and Steal
DSMZ offered to do for free
ATCC is doing a small number for a fee
In discussions with other PCC and other collections

Microorganisms Quantification gel of the genomic DNA isolated from Conexibacter woesei (DSM 14684T) Conexibacter woesei (DSM 14684T) was taken from the German Collection of Microorganisms and Cell Cultures (DSMZ). The genomic DNA was isolated using the Qiagen Genomic 500 DNA Kit (Qiagen 10262). The genomic DNA was 10-250 kb in size as determined by Pulsed Field Gel Electrophoresis (PFGE). The bulk of DNA had a size of 50-250 kb (see attached PFGE image). The DNA concentration is 500 ng/µl as estimated from the gel. Spectrophotometric measurements yielded a DNA concentration of 450 µg/ml; 300 µl of genomic DNA are shipped (150 µg). 1 2 3 4 5 6 7 8 Lane 1: c(  -Marker)= 15 ng Lane 2: c(  -Marker)= 30 ng Lane 3: c(  -Marker)= 50 ng Lane 4: DNA Molecular Weight Marker II (Roche 236250) Lane 5: DSM 13279, Collinsella stercoris Lane 6: DSM 43043, Intrasporangium calvum Lane 7: DSM 18053, Dyadobacter fermentans Lane 8: DSM 20476, Slackia heliotrinireducens Lane 9: DSM 18081, Patulibacter minatonensis Lane 10: DSM 14684, Conexibacter woesei Lane 11: DSM 11002, Dethiosulfovibrio peptidovorans Lane 12: DSM 11551, Halogeometricum borinquense Lane 13: DNA Molecular Weight Marker II (Roche 236250) Lane 14: c(  -Marker)= 125 ng Lane 15: c(  -Marker)= 250 ng Lane 16: c(  -Marker)= 500 ng 9 10 11 12 13 14 15 16

GEBA Pilot IV: Sequencing, Annotation, Data Release

Current Status
>100 in progress
GEBA 56 (focus of first paper)
34 finished genomes
55 submitted to Genbank
Released to IMG-GEBA page and JGI-FTP site
All data is completely Open for anyone to use

IMG/GEBA http://img.jgi.doe.gov/cgi-bin/geba/main.cgi

Adopt a Microbe

GEBA Pilot IV: Assess Benefits of GEBA56 All genomes have some value But what, if any, is the benefit of tree-guided sequencing over other selection methods

Why Increase Taxonomic Coverage II?
Gene discovery
Annotation, functional prediction
Metagenomic analysis
Mechanisms of diversification
Species phylogeny and classification

Value of diverse genomes I: Gene discovery
Premise:
New genomes frequently contain genetic novelty
Phylogenetic diversity of a genome should be correlated to novelty
Caveat:
Does lateral gene transfer wipe out contribution of phylogenetic diversity to novelty?

Protein Family Rarefaction Curves
Take data set of multiple complete genomes
Identify all protein families using MCL
Plot # of genomes vs. # of protein families

Genome Number Total Gene Number Number of proteins 0 50000 100000 150000 200000 250000 300000 350000 0 10 20 30 40 50 60 70 80 S. agalactiae Enterobacteriaceae Actinobacteria Bacteria from GEBA project

Novelty 2 - Structural Novelty
Of the 17000 protein families in the GEBA56, 1800 are novel in sequence (Wu)
Structural modeling suggests many are structurally novel too (D'haeseleer)
372 being crystallized by the PSI (Kerfeld)

Novelty 3 Diversity within known families

Transporter Profiles Sebaldella termitidis ATCC 33386 has 2x number of sugar PTS transporters of any genome

Novelty 4 Unusual distribution patterns

Shotgun Sequencing Detects More Diversity than PCR-methods

First Bacterial Actin Related Protein First found by V. Kunin, Structure Analysis by Patrik D. et al

Most Closely Related to ARP8

Value of 100 diverse genomes II: Annotation
Premise:
Increased phylogenetic coverage should improve our ability to annotate genes in other (e.g., reference/model genomes)

Annotation Improves
Conversion of hypothetical into conserved hypotheticals
Linking distantly related members of protein families
Non-homology functional prediction methods

Linking Protein Families Improved

Fusion Based Predictions Improved

Improving Rosetta Stone Predictions

Value of 100 diverse genomes III: Metagenomics
Premise:
Increased sampling of diverse genomes should improve many aspects of metagenomic analysis
To test:
Annotation
Binning

Metagenomic Annotation Improves (Slightly)

Compositional Binning Improves (Slightly)

Phylogenetic Binning Improves Slightly

Value of 100 diverse genomes V: Phylogeny

16s Says Hyphomonas is in Rhodobacteriales Badger et al. 2005

WGT Says Its Related to Caulobacterales Badger et al. 2005

GEBA - After the Pilot

PD of sequenced organisms

PD with GEBA

Most phyla with cultured species are sparsely sampled
Lineages with no cultured taxa even more poorly sampled
Well sampled phyla Poorly sampled No cultured taxa Proteobacteria Chlorobi Firmicutes Fusobacteria Actinobacteria Cyanobacteria Chlamydia Spriochaetes Deinococcus-Thermus Aquificae Thermotogae TM6 OS-K Termite Group OP8 Marine GroupA WS3 OP9 NKB19 OP3 OP10 TM7 OP1 OP11 Nitrospira Synergistes Deferribacteres Thermudesulfobacteria Chrysiogenetes Thermomicrobia Dictyoglomus Coprothmermobacter

Same trend in Viruses

Same trend in Microbial Eukaryotes

0.1 Acidobacteria Bacteroides Fibrobacteres Gemmimonas Verrucomicrobia Planctomycetes Chloroflexi Tree based on Hugenholtz (2002) with some modifications. Need experimental studies from across the tree too Proteobacteria Chlorobi Firmicutes Fusobacteria Actinobacteria Cyanobacteria Chlamydia Spriochaetes Deinococcus-Thermus Aquificae Thermotogae TM6 OS-K Termite Group OP8 Marine GroupA WS3 OP9 NKB19 OP3 OP10 TM7 OP1 OP11 Nitrospira Synergistes Deferribacteres Thermudesulfobacteria Chrysiogenetes Thermomicrobia Dictyoglomus Coprothmermobacter

MICROBES

A Happy Tree of Life

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Eisen.Geba.Jgi2009b

by Jonathan Eisen

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